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Wolf-Rüdiger Scheible

Publications and source records attributed to Wolf-Rüdiger Scheible.

15 recordsLinked to original sources
PubMed2007

Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon-deprived Arabidopsis seedlings.

Arabidopsis seedlings were subjected to 2 days of carbon starvation, and then resupplied with 15 mm sucrose. The transcriptional and metabolic response was analyzed using ATH1 arrays, real-time quantitative (q)RT-PCR analysis of >2000 transcription regulators, robotized assays of enzymes from central metabolism and metabolite profiling. Sucrose led within 30 min to greater than threefold changes of the transcript levels for >100 genes, including 20 transcription regulators, 15 ubiquitin-targeting proteins, four trehalose phosphate synthases, autophagy protein 8e, several glutaredoxins and many genes of unknown function. Most of these genes respond to changes of endogenous sugars in Arabidopsis rosettes, making them excellent candidates for upstream components of sugar signaling pathways. Some respond during diurnal cycles, consistent with them acting in signaling pathways that balance the supply and utilization of carbon in normal growth conditions. By 3 h, transcript levels change for >1700 genes. This includes a coordinated induction of genes involved in carbohydrate synthesis, glycolysis, respiration, amino acid and nucleotide synthesis, DNA, RNA and protein synthesis and protein folding, and repression of genes involved in amino acid and lipid catabolism, photosynthesis and chloroplast protein synthesis and folding. The changes of transcripts are followed by a delayed activation of central metabolic pathways and growth processes, which use intermediates from these pathways. Sucrose and reducing sugars accumulate during the first 3-8 h, and starch for 24 h, showing that there is a delay until carbon utilization for growth recommences. Gradual changes of enzyme activities and metabolites are found for many metabolic pathways, including glycolysis, nitrate assimilation, the shikimate pathway and myoinositol, proline and fatty acid metabolism. After 3-8 h, there is a decrease of amino acids, followed by a gradual increase of protein.

Arabidopsis↗
PubMed2007

Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus.

Affymetrix ATH1 arrays, large-scale real-time reverse transcription PCR of approximately 2200 transcription factor genes and other gene families, and analyses of metabolites and enzyme activities were used to investigate the response of Arabidopsis to phosphate (Pi) deprivation and re-supply. Transcript data were analysed with MapMan software to identify coordinated, system-wide changes in metabolism and other cellular processes. Phosphorus (P) deprivation led to induction or repression of > 1000 genes involved in many processes. A subset, including the induction of genes involved in P uptake, the mobilization of organic Pi, the conversion of phosphorylated glycolytic intermediates to carbohydrates and organic acids, the replacement of P-containing phospholipids with galactolipids and the repression of genes involved in nucleotide/nucleic acid synthesis, was reversed within 3 h after Pi re-supply. Analyses of 22 enzyme activities revealed that changes in transcript levels often, but not always, led to changes in the activities of the encoded enzymes in P-deprived plants. Analyses of metabolites confirmed that P deprivation leads to a shift towards the accumulation of carbohydrates, organic acids and amino acids, and that Pi re-supply leads to use of the latter. P-deprived plants also showed large changes in the expression of many genes involved in, for example, secondary metabolism and photosynthesis. These changes were not reversed rapidly upon Pi re-supply and were probably secondary in origin. Differentially expressed and highly P-specific putative regulator genes were identified that presumably play central roles in coordinating the complex responses of plants to changes in P nutrition. The specific responses to Pi differ markedly from those found for nitrate, whereas the long-term responses during P and N deprivation share common and non-specific features.

Arabidopsis↗
PubMed2006

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana.

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.

Arabidopsis↗
PubMed2006

PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants.

Inorganic phosphate (Pi)-signaling pathways in plants are still largely unknown. The Arabidopsis (Arabidopsis thaliana) pho2 mutant overaccumulates Pi in leaves in Pi-replete conditions. Micrografting revealed that a pho2 root genotype is sufficient to yield leaf Pi accumulation. In pho2 mutants, Pi does not repress a set of Pi starvation-induced genes, including AtIPS1, AT4, and Pi transporters Pht1;8 and Pht1;9. Map-based cloning identified PHO2 as At2g33770, an unusual E2 conjugase gene. It was recently shown that Pi deprivation induces mature microRNA (miRNA [miR399]) and that overexpression of miR399 in Pi-replete conditions represses E2 conjugase expression and leads to high leaf Pi concentrations, thus phenocopying pho2. We show here that miR399 primary transcripts are also strongly induced by low Pi and rapidly repressed after addition of Pi. PHO2 transcripts change reciprocally to miR399 transcripts in Pi-deprived plants and in miR399 overexpressers. However, responses after Pi readdition and in beta-glucuronidase reporter lines suggest that PHO2 expression is also regulated by Pi in a manner unrelated to miR399-mediated transcript cleavage. Expression of miR399 was strongly reduced in Pi-deprived Arabidopsis phr1 mutants, and a subset of Pi-responsive genes repressed in Pi-deprived phr1 mutants was up-regulated in Pi-replete pho2 mutants. This places miR399 and PHO2 in a branch of the Pi-signaling network downstream of PHR1. Finally, putative PHO2 orthologs containing five miR399-binding sites in their 5'-untranslated regions were identified in other higher plants, and Pi-dependent miR399 expression was demonstrated in rice (Oryza sativa), suggesting a conserved regulatory mechanism.

Arabidopsis↗
PubMed2005

Sugars and circadian regulation make major contributions to the global regulation of diurnal gene expression in Arabidopsis.

The diurnal cycle strongly influences many plant metabolic and physiological processes. Arabidopsis thaliana rosettes were harvested six times during 12-h-light/12-h-dark treatments to investigate changes in gene expression using ATH1 arrays. Diagnostic gene sets were identified from published or in-house expression profiles of the response to light, sugar, nitrogen, and water deficit in seedlings and 4 h of darkness or illumination at ambient or compensation point [CO(2)]. Many sugar-responsive genes showed large diurnal expression changes, whose timing matched that of the diurnal changes of sugars. A set of circadian-regulated genes also showed large diurnal changes in expression. Comparison of published results from a free-running cycle with the diurnal changes in Columbia-0 (Col-0) and the starchless phosphoglucomutase (pgm) mutant indicated that sugars modify the expression of up to half of the clock-regulated genes. Principle component analysis identified genes that make large contributions to diurnal changes and confirmed that sugar and circadian regulation are the major inputs in Col-0 but that sugars dominate the response in pgm. Most of the changes in pgm are triggered by low sugar levels during the night rather than high levels in the light, highlighting the importance of responses to low sugar in diurnal gene regulation. We identified a set of candidate regulatory genes that show robust responses to alterations in sugar levels and change markedly during the diurnal cycle.

Arabidopsis↗
PubMed2005

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression.

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NAC TF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor- and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Arabidopsis↗
PubMed2005

Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses.

MapMan is a user-driven tool that displays large genomics datasets onto diagrams of metabolic pathways or other processes. Here, we present new developments, including improvements of the gene assignments and the user interface, a strategy to visualize multilayered datasets, the incorporation of statistics packages, and extensions of the software to incorporate more biological information including visualization of corresponding genes and horizontal searches for similar global responses across large numbers of arrays.

Genes, Plant↗
PubMed2005

Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis.

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future.

Arabidopsis↗
PubMed2004

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion.

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.

Amino Acid Sequence↗
PubMed2004

Glycosyltransferases and cell wall biosynthesis: novel players and insights.

Plants need an enormous biosynthetic machinery to synthesize the complex polysaccharides that are present in the plant cell wall. The isolation, characterization and mapping of wall mutants, together with biochemical approaches, have led to significant advances in our understanding of both wall polysaccharide synthesis at a molecular level and the function of polysaccharides in plant growth and development. Moreover, potential regulation mechanisms and associated protein factors are emerging from recent data.

Arabidopsis↗
PubMed2004

Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen.

Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.

Arabidopsis↗
PubMed2004

Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes.

Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real-time RT-PCR revealed 35 root-specific and 52 shoot-specific TF genes, most of which have not been identified as organ-specific previously. Finally, many of the TF transcripts detected by RT-PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.

Arabidopsis↗
PubMed2003

An Arabidopsis mutant resistant to thaxtomin A, a cellulose synthesis inhibitor from Streptomyces species.

Thaxtomin A is a phytotoxin produced by Streptomyces scabies and other Streptomyces species, the causative agents of common scab disease in potato and other taproot crops. At nanomolar concentrations, thaxtomin causes dramatic cell swelling, reduced seedling growth, and inhibition of cellulose synthesis in Arabidopsis. We identified a mutant of Arabidopsis, designated txr1, that exhibits increased resistance to thaxtomin as a result of a decrease in the rate of toxin uptake. The TXR1 gene was identified by map-based cloning and found to encode a novel, small protein with no apparent motifs or organelle-targeting signals. The protein, which has homologs in all fully sequenced eukaryotic genomes, is expressed in all tissues and during all developmental stages analyzed. Microarray transcript profiling of some 14,300 genes revealed two stomatin-like genes that were expressed differentially in the txr1 mutant and the wild type. We propose that TXR1 is a regulator of a transport mechanism.

Amino Acid Sequence↗
PubMed2002

Steps towards an integrated view of nitrogen metabolism.

This article discusses how nitrate assimilation is integrated with nitrate uptake, with ammonium assimilation and amino acid synthesis, with pH regulation, and with the sugar supply in tobacco leaves. During the first part of the light period, nitrate assimilation exceeds nitrate uptake by 2-fold and ammonium assimilation by 50%, leading to rapid depletion of nitrate and accumulation of ammonium, glutamine, glycine and serine. NIA, NII and PPC expression show a shared maximum early in the diurnal cycle to direct carbon towards malate synthesis for pH regulation. Later in the diurnal cycle an orchestrated increase of GLN2, PKc, CS, and ICDH-1 expression re-establishes a balance between nitrate assimilation and ammonium metabolism. Nitrate uptake continues throughout the night, replenishing the leaf nitrate pool. These diurnal changes are attenuated or abolished in mutants with low NIA activity, and modified in wild-type plants growing on different nitrogen sources or elevated [CO(2)]. Comparison across genotypes and conditions reveals that NIA transcript levels are always closely related to the balance between nitrate influx and assimilation, but are unrelated to changes of glutamine or 2-oxoglutarate. In a systematic search for other downstream regulators, a wide range of downstream metabolites was fed to detached leaves and glutamate, cysteine, asparagine, and malate identified as candidates. Low sugars totally inhibit nitrate assimilation, overriding signals from nitrogen metabolism. Moderate changes act post-transcriptionally, and larger changes lead to a collapse of the NIA transcript. Low sugars also lead to a collapse of minor amino acids and a dramatic decrease of phenylpropanoids and nicotine. Consequently, wild-type plants growing in unfavourable light regimes and antisense RBCS transformants are simultaneously carbon- and nitrogen-limited.

Amino Acid Oxidoreductases↗